Project description:Ribosome profiling (Ribo-Seq) and RNA-Seq analysis of eEF3 depletion in yeast (Saccharomyces cerevisiae). eEF3 depletion was induced by methionine in a modified strain where the native promoter was replaced by methionine repressible MET25 promoter. Conditional depletion enables us to study global effects of an essential gene.
Project description:Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from storage carbohydrates were 2-3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae. The yeast was first grown for 14 days under extreme calorie restriction in anaerobic, glucose-limited retentostats (Boender et al., 2009, Appl.Environ.Microbiol., 75: 5607-5614.). Subsequently, starvation was started by terminating the glucose feed. Yeast transcriptional reprogramming in response to calorie restriction and starvation was monitored by microarray analysis. Independent duplicate retentostat cultures, and subsequently starvation, were sampled for transcriptome analysis using Affymetrix microarrays. One time-point was sampled during calorie restriction (T0) and four time points were sampled during the starvation phase 10, 30, 60 and 120 minutes after switching of the feed, resulting in a dataset of 10 arrays.
Project description:In our previous work, we showed the positive effect of the magnesium and the negative effect of the copper on yeast fermentation performance. The magnesium increases the ethanol yield and a faster glucose consumption by the yeast, on the other hand, the copper provides an opposite effect in yeast under fermentation condition. Therefore, from this contrasting effect we performed the gene-wide expression analysis in the industrial yeast Saccharomyces cerevisiae JP1 under fermentation condition in order to reveal the gene expression profile upon magnesium and copper supplementation.
Project description:The yeast protein kinases Sat4/Hal4 and Hal5 are required for the plasma membrane stability of the K+ transporter Trk1 and some amino acid and glucose permeases. The transcriptomic analysis presented here indicates alterations in the general control of both nitrogen and carbon metabolism. Accordingly, we observed reduced uptake of methionine and leucine in the hal4 hal5 mutant. This decrease correlates with activation of the Gcn2-Gcn4 pathway, as measured by expression of the lacZ gene under the control of the Gcn4 promoter. However, with the exception of methionine biosynthetic genes, few amino acid biosynthetic genes are induced in the hal4 hal5 mutant, whereas several genes involved in amino acid catabolism are repressed. Concerning glucose metabolism, we found that this mutant exhibits derepression of respiratory genes in the presence of glucose, leading to an increased activity of mitochondrial enzymes, as measured by SDH activity. In addition, the reduced glucose consumption in the hal4 hal5 mutant correlates with a more acidic intracellular pH and with low activity of the plasma membrane H+-ATPase. As a compensatory mechanism for the low glycolytic rate, the hal4 hal5 mutant overexpresses the HXT4 high affinity glucose transporter and the hexokinase genes. These results indicate that the hal4 hal5 mutant presents defects in the general control of nitrogen and carbon metabolism, which correlate with reduced transport of amino acids and glucose, respectively. A more acidic intracellular pH may contribute to some defects of this mutant. Four biological replicates were used to assess diferentially expression between wild type yeast strain and the hal4hal5 mutant strain for three sample sets: 1) cells grown in YPD pH 4.5 and BY4741 genetic background, 2) cells grown in YPD pH 4.5 and W303 genetic background, 3) cells grown in YPD pH 6.0 and W303 genetic background. Differentially expressed genes were identified using one-class significant analysis of microarrays (SAM; Tusher et al, 2004)
Project description:The canonical role of eEF1A is to deliver the aminoacyl tRNA to the ribosome, we have used the yeast model system to investigate further roles for this protein. We used microarray to study the transcriptomic effects of elevated levels of eEF1A on yeast cells during log phase growth